Osteoinductive biomaterials

ABSTRACT

In a method of isolating osteogenic protein from bone, in which an osteogenic protein-containing fraction is extracted from bone and enriched by a sequence of enrichment steps selected from ultrafiltration and chromatography, the invention provides the improvement of removing higher molecular weight components from the osteogenic protein-containing fraction prior to the enrichment steps. The higher molecular weight components have a molecular weight of about 100-300 kDa and are selected from collagen, collagen fragments, collagen aggregates and mixtures thereof.

THIS INVENTION relates to osteoinductive biomaterials. In particular theinvention relates to an osteogenic composition and to the use of anosteogenic composition in therapy.

The osteogenic composition of the invention is particularly intended forhuman or mammalian tissue regeneration and for promoting or inducingbone growth. For the purposes of this specification, the phrase“osteogenic protein” refers to the material which is obtained byfractionation of total mammalian bone protein and which is capable ofinducing bone formation. The terms “osteogenic” and “osteoinductive” areconsidered to be synonymous. Osteogenesis is the term used to describethe de novo formation of bone in adult mammals and is evidenced inadults during regeneration of bone fractures. It proceeds via a processwhich closely resembles embryonic osteogenesis. Osteogenic proteincontains, amongst other unidentified proteins, Bone MorphogeneticProteins (BMPs). This is a family of characterized proteins which havebeen classified as part of the larger transforming growth factor-betasuperfamily of morphogenic proteins. The BMP family comprises more thana dozen individual members which are known to be capable of inducingbone formation in mammals.

According to a first aspect of the invention, in a method of isolatingosteogenic protein from bone, in which an osteogenic protein containingfraction is extracted from bone and enriched by a sequence of enrichmentsteps selected from ultrafiltration and chromatography, there isprovided the improvement of removing higher molecular weight componentsfrom the osteogenic protein containing fraction prior to the enrichmentsteps.

The higher molecular weight components may have a molecular weight ofabout 100-300 kDa. The higher molecular weight components will typicallyinclude collagen, collagen fragments, collagen aggregates and mixturesthereof.

The method may include removing the higher molecular weight componentsby ultrafiltration. For example, the components may be removed byultrafiltration through a 100-300 kDa nominal molecular weight membranesuch as a 100-300 kDa nominal molecular weight polysulphone membrane.

The osteogenic protein containing fraction may be extracted from thebone using a chaotropic solution. The chaotropic solution may containurea, guanidinium chloride or combinations thereof.

The enrichment steps may include successive ultra-filtration of theosteogenic protein containing fraction through progressively smallernominal molecular weight membranes followed by, or interspersed with,chromatographic enrichment steps. The enrichment steps may thus includeone or more chromatographic enrichment steps.

The osteogenic protein containing fraction may be concentrated anddesalted through successive ultra-filtration steps. The fraction may beconcentrated and desalted through 10 kDA and 5 kDA ultra-filtrationsteps.

The chromatographic enrichment steps may be selected from one or more ofheparin-sepharose chromatography, hydroxyapatite chromatography,reverse-phase silica chromatography and combinations of any two or morethereof.

According to a second aspect of the invention, there is provided a bonegrowth inducing composition which includes osteogenic protein, insolublebone matrix (ICBM) and gelatin.

The composition may be in the form of a hydratable powder.

The insoluble bone matrix may be prepared by demineralizing whole bonepowder with acid to produce an acid demineralised whole bone powder ormatrix, extracting soluble components from the demineralised bone powderor matrix with a chaotropic agent such as aqueous urea or a guanidiniumsolution, water-washing the residue and then drying the residue toproduce insoluble bone matrix.

The gelatin may be obtained by extracting insoluble bone matrix toproduce a fraction rich in soluble collagen type I, precipitating thesoluble collagen type I and drying the precipitate in vacuo.

The extraction may be with purified boiling water and the precipitationmay be with ethanol.

The insoluble collagenous bone matrix may be mammalian. It may benon-human or human insoluble collagenous bone matrix. It is preferablyhuman insoluble collagenous bone matrix or hICBM. The gelatin mayaccordingly be human gelatin.

The osteogenic protein may be prepared by a method as hereinbeforedescribed.

The mass ratio between the osteogenic protein, the hICBM and the humangelatin may be about 0.4-0.6: 800-1200: 100-1000.

Thus, the bone growth inducing composition may include the osteogenicprotein in an amount of about 400-600 μg, the hICBM in an amount ofabout 800-1200 mg and the human gelatin in an amount of about 100-1000mg. In a preferred embodiment, the bone growth inducing compositionincludes the osteogenic protein in an amount of about 500 μg, the hICBMin an amount of about 1000 mg and the human gelatin in an amount ofabout 200 mg.

The invention extends to a bone growth inducing composition as describedabove in which the osteogenic protein is prepared by an improved methodas described above.

According to another aspect of the invention, there is provided a methodof preparing a bone growth inducing composition, the method includingthe steps of combining osteogenic protein, insoluble bone matrix andgelatin.

The insoluble collagenous bone matrix may be mammalian and may beselected from non-human and human insoluble collagenous bone matrix. Itis preferably human insoluble collagenous bone matrix or hICBM. Thegelatin may be human gelatin.

The osteogenic protein may be prepared by a method as hereinbeforedescribed.

The osteogenic protein, the hICBM and the human gelatin may be combinedin a mass ratio of 0.4-0.6: 800 :1200: 100-1000.

The method may thus include combining the osteogenic protein in anamount of about 400-600 μg, the hICBM in an amount of about 800-1200 mgand the human gelatin in an amount of about 100-1000 mg. In a preferredembodiment, the method may include combining the osteogenic protein inan amount of about 500 μg, the hICBM in an amount of about 1000mg andthe human gelatin in an amount of about 200 mg.

The method may include combining the osteogenic protein with the hICBMin a dilute aqueous acidic solution, lyophilising the resulting mixtureto produce a dry powder and mixing the powder with the human gelatin toproduce a hydratable material.

According to another aspect of the invention, there is provided a devicefor inducing bone growth in a mammal, the device including a bone growthinducing composition which comprises osteogenic protein, insoluble bonematrix and gelatin and a delivery mechanism for delivery of thecomposition to a treatment site.

The osteogenic protein, the insoluble bone matrix and the gelatin may beas hereinbefore described. The delivery mechanism may be a syringe.

In particular, the composition may be the hydratable powder hereinbeforedescribed which may be contained in the syringe. Thus, by drawing anaqueous saline solution up into the syringe, the material may behydrated for injection into the delivery site.

According to another aspect of the invention, there is provided a methodof inducing bone formation in a mammal having a skeletal defect, themethod including the step of implanting a bone inducing composition ashereinbefore described into the skeletal defect of the mammal.

According to another aspect of the invention, there is provided a methodof inducing the growth of ectopic bone in a mammal, the method includingthe step of implanting a bone inducing composition as hereinbeforedescribed in a non-bony site of the mammal.

According to another aspect of the invention, there is provided a methodof accelerating allogeneic bone healing in a mammal, the methodincluding the step of implanting allogeneic bone material together witha bone inducing composition as hereinbefore described into site in whichallogeneic bone healing in the mammal is required.

The allogeneic bone material may be tissue-banked bone including humancortical bone chips, cancellous bone blocks, cancellous bone powder,whole bone or demineralised bone matrix.

According to another aspect of the invention, there is provided a methodof accelerating autogenous bone graft healing in a mammal, the methodincluding the step of implanting autogenous bone material together witha bone inducing composition as hereinbefore described into site in whichautogenous bone graft healing in the mammal is required.

The autogenous bone material may be iliac crest autogenous bone.

According to another aspect of the invention, there is provided asubstance or composition for use in a method of inducing bone formationin a mammal having a skeletal defect, the substance or compositioncomprising a bone growth inducing composition as hereinbefore describedand the method including implanting the composition into a skeletaldefect of the mammal.

According to another aspect of the invention, there is provided asubstance or composition for use in a method of inducing the growth ofectopic bone in a mammal, the substance or composition comprising a bonegrowth inducing composition as hereinbefore described and the methodincluding the step of implanting the composition in a non-bony site ofthe mammal.

According to another aspect of the invention, there is provided asubstance or composition for accelerating allogeneic bone healing in amammal, the substance or composition comprising a bone growth inducingcomposition as hereinbefore described and the method including the stepof implanting allogeneic bone material together with the compositioninto a site in which allogeneic bone healing in the mammal is required.

The allogeneic bone material may be tissue-banked bone selected fromhuman cortical bone chips, cancellous bone blocks, cancellous bonepowder, whole morselised bone or demineralised bone matrix.

According to another aspect of the invention, there is provided asubstance or composition for accelerating autogenous bone graft healingin a mammal, the substance or composition comprising a bone growthinducing composition as hereinbefore described and the method includingthe step of implanting autogenous bone material together with thecomposition into a site in which autogenous bone graft healing in themammal is required.

The autogenous bone material may be morselised iliac crest autogenousbone.

According to another aspect of the invention, there is provided the useof a substance or composition in the preparation of a medicament for usein a method of inducing bone formation in a mammal having a skeletaldefect, the substance or composition comprising a bone growth inducingcomposition as hereinbefore described.

According to another aspect of the invention, there is provided the useof a substance or composition for the preparation of a medicament foruse in a method of inducing the growth of ectopic bone in a mammal, thesubstance or composition comprising a bone growth inducing compositionas hereinbefore described.

According to another aspect of the invention, there is provided the useof a substance or composition in the preparation of a medicament foraccelerating allogeneic bone healing in a mammal, the substance orcomposition comprising a bone growth inducing composition ashereinbefore described and an allogeneic bone material.

The allogeneic bone material may be tissue-banked bone selected fromhuman cortical bone chips, cancellous bone blocks, cancellous bonepowder, whole bone or demineralised bone matrix.

According to another aspect of the invention, there is provided the useof a substance or composition in the preparation of a medicament foraccelerating autogenous bone graft healing in a mammal, the substance orcomposition comprising a bone growth inducing composition ashereinbefore described and an autogenous bone material.

The autogenous bone material may be morselised iliac crest autogenousbone.

This invention accordingly relates to the preparation of osteogenicprotein from mammalian bone and to its use in conjunction with a matrixin bone repair.

DISCUSSION

Mammalian bone tissue is host to a family of protein growth anddifferentiation factors, called the bone morphogenetic proteins (BMPs).These proteins are capable of inducing new bone formation when implantedin adolescent and adult mammals.

The BMPs are redeployed in adults to cause regeneration of bone viamechanisms closely resembling embryonic differentiation. Thedevelopmental cascade of bone differentiation consists of chemotaxis ofmesenchymal cells, proliferation of progenitor cells, differentiation ofcartilage, vascular invasion, bone formation, remodeling, and finallymarrow differentiation (Reddi, (1981) Collagen Rel. Res. 1:209-226). Ithas been shown that the natural endochondral bone differentiationactivity of bone matrix can be dissociatively extracted andreconstituted with inactive residual matrix to restore full boneinductive activity (Sampath and Reddi, (1981) Proc. Natl. Acad. Sci. USA78:7599-7603). The purification of osteogenin, an osteogenic proteinfrom mammalian bone is disclosed by Sampath et al. (1987) (Proc. Natl.Acad. Sci. USA 84, 7109-7113. Urist et al. (Proc. Natl. Acad. Sci. USA(1984) 81:371-375) disclose a bovine bone morphogenetic protein extracthaving the properties of an acidic polypeptide and a molecular weight ofapproximately 18 kD. The authors report that the protein is present in afraction separated by hydroxyapatite chromatography, and that it inducesbone formation in mouse hindquarter muscle and bone regeneration intrephine defects in rat and dog skulls.

European Patent Application No.148,155, published Oct. 7, 1985,discloses osteogenic proteins derived from bovine, porcine, and humanorigin. One of the proteins, designated by the inventors as a P3 proteinand having a molecular weight of 22-24 kD, is reported to have beenpurified to an essentially homogeneous state. This material is reportedto induce bone formation when implanted into animals.

It is an object of the invention to provide a method for the preparationof osteogenic protein from mammalian bone tissue in high yield. Anotherobject is to provide bone-inducing devices comprising osteogenic proteinadsorbed onto matrix as a delivery system for said bone morphogeneticproteins.

The invention provides osteogenic devices which, when implanted at askeletal defect site of the mammal, induce at the site of implantationthe full regeneration of bone and the consequent healing of the defect.The device comprises a matrix carrier material, as described below, andosteogenic protein, a fraction of total extractable bone protein whichcontains bone morphogenetic proteins (BMPs).

Osteogenic protein requires the presence of a suitable delivery materialto exert its bone regenerating effects. Matrix purified fromdemineralised bone matrix is such a suitable material and is describedin more detail below.

The method used to isolate osteogenic protein exploits in part, thepublished procedure of Sampath et al. (1987) (Proc. Natl. Acad. Sci. USA84, 7109-7113. This procedure exploits the BMPs' affinity for heparinand hydroxyapatite immobilized onto chromatographic support matrices toachieve isolation of BMP rich fractions. The procedure entails thechromatography of urea extracts-of demineralised bone onto a heparinchromatography column, followed by a hydroxyapatite column, and finallygel exclusion chromatography to eliminate heavy molecular weightcontaminants. Although this procedure results in the effective isolationof a fraction with osteogenic capacity, the quantity, yield and speed ofpurification of osteogenic protein using the method of the presentinvention is greatly improved.

The preparation of the osteogenic protein of the invention is based onthe procedure of Sampath et al (1987), but includes a novel andinventive modification.

The key modification involves the fractionation of the total boneprotein extract into a high and a low molecular weight fraction at thebeginning of the purification process, before chromatography. The bonemorphogenetic proteins have a molecular weight of approximately 30 kDaand, for the purposes of this specification, are classed as lowmolecular weight polypeptides. For the purposes of this specification,high molecular weight polypeptides include polypeptides with a molecularweight greater that 100 kDa and especially greater that 300 kDa. Thehigh molecular weight fraction will include collagen and collagenfragments (approximately 100 kDa) as well as collagen aggregates (200kDa and greater) and other unidentified polypeptides, some of which arethought to be inhibitors of morphogen-induced osteogenesis. It isimportant to note, for the purposes of this specification, thatcollagens are separated from the low molecular weight fraction at thebeginning of the process before the heparin affinity chromatographystep.

The removal of collagen is important for the following reasons. Firstly,collagen type I is known to have an affinity for BMPs (Reddi AH (1995)Cartilage morphogenesis: role of bone and cartilage morphogeneticproteins, homeobox genes and extracellular matrix. Matrix Biol. October14(8):599-606.; Winn S R, Uludag H, Hollinger J O. (1999) Carriersystems for bone morphogenetic proteins. Clin Orthop 1999 October (367Suppl):S95-106). Secondly, peptides are large MW peptides which tend tofoul columns and alter the exchange dynamics of the BMP with the bindingsites on the heparin molecule in a way which hampers binding.Furthermore, it appears that there may exist inhibitors of bonemorphogenetic proteins, the active constituent of osteogenic protein,that reside in the high molecular weight fraction of total extractablebone protein. This implies that there exists competitive binding forBMPs between collagen type I and heparin. This competitive bindinginterference appears to result in yield losses during thechromatographic purification of BMPs on a heparin column. The method ofthis invention results in a significant improvement in the recovery oftotal osteogenic activity over prior art methods.

The invention is now described, by way of example with reference to thefollowing Example and the Figure in which

FIG. 1 shows X-ray evaluation scores of treated non-unions as a functionof time;

FIG. 2 shows non-union in a bone of a patient after conventionaltreatment; and

FIG. 3 shows complete healing of the bone of the patient of FIG. 2 aftertreatment in accordance with the method of the invention

EXAMPLE 1

Purification of Human Osteogenic Protein Containing Bone MorphogeneticProteins

1. Preparation of Demineralised Bone

Human long bone diaphyses, freed from adhering soft tissues, weredemarrowed and cut into pieces of between 1 and 4 cm. Batches of thismaterial were defatted in a solvent system comprising a 50:50 ratio byvolume of methanol and chloroform, at 4° C.-8° C. for 16-24 hours. Thebone was then dehydrated in absolute alcohol for 48 hours at 4° C.-8° C.The alcohol was decanted and the bone was air-dried in a fume hood for48-96 hours. The bone was then milled in a hammer mill to a particlesize ranging from 10 to 425 micron.

The particulate material was demineralised at room temperature, withconsecutive additions of four to five volumes of 0.5 M HCl, until acidbase reaction between the hydroxyapatite of the bone and the HCl hadneared completion as judged by slowing pH changes over time. Thedemineralised bone was neutralized with dilute sodium bicarbonatesolution, and washed with purified water to produce demineralised bonematrix.

2. Dissociative Extraction of Demineralised Bone Matrix

Demineralised bone matrix (DBM) from the previous step was extractedtwice with three to four volumes of 8M urea, 50 mM Tris-HCl, pH 7.4,containing protease inhibitors (5 mM benzamidine hydrochloride, 0.1 M6-aminohexanoic acid, 5 mM N-ethylmaleimide and 0.5 mMphenylmethylsulfonyfluoride) for 24 hours at 4° C. to 8° C. Thesupernatant was collected by filtration through a porous polypropylenefrit, and filtered through a three micron nominal size cartridge filter(Polygard, Millipore Corporation, USA).

3. Ultrafiltration Fractionation of High Molecular Weight Components

Heavy molecular proteins and collagens were removed by ultrafiltrationof the supernatant from the step 2 through a polysulfone 300 kDa nominalmolecular 20 weight membrane (Millipore, Cat. No. CDUF006TM). A 100 kDanominal molecular weight membrane can optionally be employed withsomewhat lower yields of total BMP activity, but with higher specificactivity. This procedure removed collagens, especially type I collagens,which bind BMPs under conditions of lower ionic strength. The retentatewas washed a few times with 6 M urea buffer, 50 mM Tris-HCL pH 7.4(Buffer A), and the diafiltrate which contained the osteogenic activitywas collected.

4. Concentration and Desalting by Ultrafiltration Buffer Exchange

The diafiltrate containing the osteogenic proteins (molecular weightcirca 30 kDa) from step 3 was desalted and concentrated byultrafiltration on a 10 kDa PLGC membrane (Millipore, Cat. No.SK1P003W4). This step effectively removed salt and other low MW weightcomponents, to create the required conditions for the followingchromatographic step. Successive volumes of Buffer A containing theaforementioned concentrations of enzyme inhibitors but excluding n-ethylmaleimide were added to the retentate following concentration, until theconductivity of the retentate reached between 5.0 and 6.0 milli Siemens.

5. Heparin-Sepharose Chromatography

The retentate from step 4 was chromatographed onto Heparin-SephahroseCL-6b (Pharmacia-Amersham) which had been equilibrated with buffer Acontaining 0.15 M NaCl. The column was washed with three column volumesof buffer A containing 0.15 NaCl and then eluted with buffer Acontaining 0.5 M NaCl. The eluting peak with absorbance at 280 nm wascollected and stored at 4° C.

6. Ultrafiltration Exchange of Heparin-Sepharose Affinity Fraction

The Heparin-Sepharose affinity fraction from step 5 was desalted andconcentrated by ultrafiltration on a PLCC 5 kDa membrane (Millipore,Cat. No. CDUF001LC). This step effectively removed salt and other low MWweight components, to create the required conditions for the nextchromatographic step. Successive volumes of Buffer A containing 10 mMsodium phosphate were added to the retentate following concentration,until the conductivity of the retentate had reached between 5.0 and 6.0milli Siemens.

7. Hydroxyapatite (HA) Chromatography

The retentate from step 6 was chromatographed onto a hydroxyapatitecolumn (Hydroxyapatite Ultrogel, Biosepra, France) which had beenequilibrated in Buffer A containing 10 mM sodium phosphate. The columnwas washed with three column volumes of Buffer A containing 10 mM sodiumphosphate. An osteogenic protein enriched fraction was eluted withBuffer A containing 150 mM sodium phosphate. The eluting peak withabsorbance at 280 nm was collected and stored at 4° C.

8. Exchange of HA Affinity Fraction into 10 mM HCl

The HA affinity fraction from step 7 was exchanged into a 10 mM HClsolution using an Amicon stirred Ultrafiltration cell (MilliporeCorporation, U.S.A.) loaded with a 3 kDa cutoff cellulose membrane (YM3,76 mm regenerated cellulose, Millipore Corporation U.S.A.).

In an embodiment of the invention, the HA affinity fraction was insteadloaded onto a C-18 Vydac silica-based HPLC column (particle size 5 um,pore size 300 A). The column was washed with 0.1% trifluoroacetic acid,10% acetronitrile for 10 column volumes, and the bound proteins werestep eluted with a 70% acetonitrile, 0.1% trifluoroacetic acid. Thismaterial was lyophilized and reconstituted into 10 mM HCl.

The process flow chart with protein values is set out in Table 1. TABLE1 Total protein Step Procedure (mg) 1 Dehydrated, defatted human bonepowder 5 Not determined kg. 2 Extraction with chaotropic solutioncontaining 17 703 urea or guanidinium chloride 3 Filtration through aMillipore Polygard CR Not determined cartridge filter with a 3.0 micronnominal pore size product no. CR0301006. 4 Ultrafiltration employing amembrane with a 15 069 nominal pore size of 300 kD 5 Ultrafiltration andbuffer exchange employing 8 881 a membrane with a nominal pore size of10 kD 6 Heparin affinity fraction 211.43 7 Hydroxyapatite affinityfraction 62.99 8 Ultrafiltration exchange or HPLC 40-60

500 μg of the material from step 8 of Example 1, delivered on 1.2 gramsof matrix, induces new bone formation in recalcitrant long-bonenon-unions in humans. The material from step 8 was analysed by S-200 gelfiltration chromatography (Pharmacia) and found to contain 20% by massof high molecular weight components. These may be optionally removed bya ‘polishing’ step employing gel exclusion chromatography on S-200matrix (Pharmacia) and elution with 8M Urea, 1 M NaCl, 50 mM Tris-HCl pH7.4. The final yield is in the region of 30 mg to 50 mg of osteogenicprotein. These yields are approximately four-fold higher than thosepreviously reported for baboon bone extraction employing a comparativestarting bone material and method on hydoxyapatite affinity, heparinaffinity and gel exclusion chromatography on S-200 matrices(Pharmacia)(Ripamonti U, Ma S S, Cunningham N S, Yeates L, Reddi A H(1993) Reconstruction of the bone-bone marrow organ by osteogenin, abone morphogenetic protein, and demineralised bone matrix in calvarialdefects of adult primates (Plastic and Reconstructive Surgery91(1):27-36). Comparative histomorphometric studies between iliac crestbone biopsies of humans and baboons have demonstrated a remarkabledegree of similarity between the two species (Schnitzler C M, RipamontiU, and Mesquita J M (1993) Histomorphometry of iliac crest trabecularbone in adult male baboons in captivity, Calcif. Tiss. Int., 52,447-454).

EXAMPLE 2 Determination of Osteogenic Activity of Osteogenic Protein

Bioassay in Rats

Osteogenic activity was bioassayed as described by Sampath and Reddi(Proc. Natl. Acad. Sci. USA (1983) 80:6591-6595). The assay consists ofimplanting test samples comprising insoluble bone matrix and humanosteogenic protein in subcutaneous sites in recipient rats under etheranesthesia. A vertical incision (1 cm) was made under sterile conditionsin the skin over the thoracic region, and bilateral pockets wereprepared by blunt dissection. Implants comprised 25 mg rat ICBM, 50 mgrat tail type I collagen in 0.5 M acetic acid, and osteogenic protein invarying amounts. The test sample was implanted bilaterally into eachpocket and the incision was closed with stitches. The heterotropic siteallowed for the study of bone induction without the possible ambiguitiesresulting from the use of bony sites.

Regenerated tissues were explanted on day 12 post-implantation andassayed for alkaline phosphatase activity, a marker for bone formation,as described (Reddi and Sullivan 1980 Endocrinology 107:1291-1299).Results and data. are presented in Table 2. TABLE 2 Rat bioassayreplicates 4 Micrograms osteogenic protein assayed per implant 100 μgAlkaline phosphatase activity units/mg protein 8.82 U/mg (average 4replicates) (sd = 3.8)*hICBM—human insoluble bone matrix.

The implant model in rats exhibited a controlled progression through thestages of osteogenic protein induced endochondral bone development. Thisposffoetal osteogenesis may be considered to recapitulate events thatoccur in the normal course of embryonic bone development. The new boneresulted from local mesenchymal condensations, a cartilage phase andextracellular matrix production, vascular invasion and mineralisation,and finally the formation of new bone via the differentiation ofosteoprogenitor cell lines.

Histological analysis employing staining with toluidine blue orhemotoxylin/eosin demonstrated clearly the development of endochondrialbone. Twelve day implants were usually sufficient to determine whetherthe implants showed bone inducing activity.

Alkaline phosphatase activity may be used as a marker for osteogenesis.The enzyme activity may be determined spectrophotometrically afterhomogenization of the implant and assaying of enyme activity with thesubstrate p-nitrophenyl phosphate under alkaline conditions. Implantsshowing no bone development by histology should have no alkalinephosphatase activity under these assay conditions (Reddi A H andSullivan N E (1980) Endocrinology 107, 1291-1299). The assay is usefulfor quantitation of the specific and total activity of alkalinephosphatase, which may then be correlated to the osteoinductive potencyof the prepared osteogenic protein described herein.

Alkaline phosphatase activity is calculated according to the method ofReddi and Sullivan (1980, Endocrinology 107, 1291-1299). Induction of 1unit or more of alkaline phosphatase by a rat implant indicateseffective osteogenesis.

EXAMPLE 3 Preparation of Human Gelatin

An amount of ICBM was combined with five to ten volumes of water in aborosilicate glass bottle and heated in a pressure cooker for one hour.The supernatant was filtered through Whatman no. 1 paper or a 20 micronstainless steel mesh. The gelatinous solution was cooled to 25° C. and 5volumes of chilled ethanol (−20° C.) were added to precipitate collagen.The precipitate was dried in vacuo, and milled to a size range of 75 to425 micron.

EXAMPLE 4 Fabrication of Osteogenic Devices

Human ICBM was used as the adsorptive carrier matrix for the fabricationof the osteoinductive composite biomaterial. Inactive ICBM was restoredto biological activity when a sufficient amount of osteogenic proteinwas combined with ICBM. The particle size of the ICBM influences thequantitative response of new bone. Particles between 75 and 420 μmelicit the maximum response. An amount of ICBM was combined withosteogenic protein in 10 mM HCl and thoroughly mixed with sterilespatula. The material was lyophilized to dryness.

This material was then combined with human ICBM-derived gelatin. Thecomponents were thoroughly dry-mixed together to obtain a homogeneouslydistributed composition. The human gelatin acted as a readily hydratablematerial that causes the biomaterial to become extrudable. The compositewas packed into a syringe. The osteoinductive composite was rehydratedat the time of use. Rehydration was achieved when an amount of sterilesaline or water was drawn into the syringe, and approximately 10 minutesallowed for rehydration to occur. The material could then easily beexpelled out of the syringe by depressing the plunger. This allowed forprecise implant deposition into a defect site at the time of surgery.The implant may be further contained in situ using standard gelatinoussponges such as Spongostan (Johnson and Johnson Medical Limited, U.K.).

The carrier could be replaced by either a biodegradable-synthetic orsynthetic-inorganic matrix (e.g., HAP, collagen, tricalcium phosphate,or polylactic acid, polyglycolic acid and various copolymers thereof).

Table 3 sets out a typical formulation for the osteogenic composition.TABLE 3 Material Amount Osteogenic protein  500 μg hICBM* 1000 mg Humangelatin  200 mg TOTAL 1200 mg*hICBM—human insoluble bone matrix.

EXAMPLE 5 Testing of Osteogenic Composition in Humans

Implants containing 500 micrograms of human osteogenic protein adsorbedonto a composite matrix comprising 1 g of insoluble bone matrix and 200mg of lyophilized human gelatin were prepared. Thirty-four patients withresistant nonunions including partial or complete segmental defects weretreated with the osteogenic composite. The series consisted of 11females and 23 males. The average age was 36 years. All patients hadpreviously been variously treated by internal or external fixation,cast, and/or autogeneic bone grafting, and failed to achieve union.Preoperative symptoms averaged 26 months (range, one to 228 months). Theimplant was incorporated at the time of surgery by injecting thehydrated implant at the defect site, which was further stabilised byinternal or external fixation. An average of 2.4 g of the composite wasused per patient. Seventeen patients additionally received supplementarybone which included cancellous bone particles and block configuredspongy bone. Functional results were rated according to weight-bearingfunction at follow-up periods of 1, 8, 16 and 24 week periodspost-operatively. A zero score was allocated where there was no weightbearing, a one score allocated for weight bearing with the assistance oftwo crutches, a two score allocated for light weight bearing with onecrutch, a three score allocated for full weight bearing with one crutchand a four score allocated for full weight bearing with no crutchassistance. The average score was 3.25 for an average follow-up time of17 weeks (range eight to 32 weeks). This score was higher than thepre-operative score of 2.22 and the post-operative score (week 1) of0.5. Of the five patients who suffered recurrent infection, two failedto score above 2 at 18.5 weeks average follow-up period. Bridging was asassessed radiographically by trained clinicians according to a scalefrom 1 to 5 where 1=non-union/no callus, 2=callus present withoutbridging, 3=moderate bridging, 4=good bridging, 5=complete union. Theaverage score for the treatment group on follow-up period of 20 weekswas 2.80 in comparison to the pre-treatment score of 1.44. The resultsindicate that the osteogenic composite implant of the invention resultsin effective treatment of difficult nonunions. The scores as measured atdifferent follow-up periods are presented in FIG. 1.

It is an advantage of the invention that the multistep developmentalcascade of bone induced by the osteogenic biomaterial composite of theinvention includes binding of fibrin and fibronectin to the biomaterial,chemotaxis of cells, proliferation of fibroblasts, mesenchymalcondensation, differentiation into chondroblasts, chondrogenesis,vascular invasion, bone formation, remodeling, and bone marrowdifferentiation.

The injectable biomaterial of the invention offers several advantages.It may be stored at room temperature for lengthy periods without markeddeterioration in biological activity. It may be readily rehydrated atthe time of surgery and it is easily handled, merely requiring thedepression of the syringe plunger to expel the osteogenic material asrequired.

From a clinical context, the osteogenic biomaterial composite offers thefollowing advantages. It is osteogenic, inducing bone at the site ofimplantation. It obviates the need to perform a second operation at thepatient's hip to harvest autologous bone and it obviates the need to usetissue banked bone. This reduces the risk of transmissible diseases.

The ICBM binds osteogenic protein and acts as a slow release deliverysystem to activate progenitor cells at the site of implantation. Thecomposite biomaterial of the invention accommodates each step of thecellular response during bone development. It is biocompatible, and isresorbed during osteogenesis and replaced by the host's own bone.

The geometry of the described biomaterial as measured by its particlesize, is optimal in permitting cell infiltration and differentiation.

1-35. (canceled)
 36. A method of preparing an osteogenic proteinfraction, comprising: extracting demineralized bone matrix with asolution of at least one chaotropic agent selected from the groupconsisting of urea and guanidinium salts to produce an extract; removinghigh molecular weight proteins which exceed 100-300 kDa from the extractby ultrafiltration to produce a lower molecular weight fraction;subjecting the lower molecular weight fraction to heparin affinitychromatography under conditions which first favor the binding and thenthe elution of a purified heparin affinity fraction containing theosteogenic protein fraction; subjecting the heparin affinity fraction tohydroxyapatite chromatography under conditions which first favor thebinding and then the elution of a purified osteogenic protein fraction;and exchanging the purified osteogenic protein fraction into a solventsuitable for human medical use.
 37. A bone growth inducing composition,comprising a combination of an osteogenic protein fraction prepared bythe method as claimed in claim 36, a carrier matrix and gelatine.
 38. Abone growth inducing composition, comprising a combination of anosteogenic protein prepared by the method of claim 36, a carrier matrixand gelatine, in order to produce a mixture, wherein the mixture islyophilized to produce a hydratable powder.
 39. The bone growth inducingcomposition as claimed in claim 38, wherein the carrier matrix isselected from the group consisting of insoluble bone matrix, abiodegradable synthetic matrix and a synthetic inorganic matrix.
 40. Thebone growth inducing composition as claimed in claim 39, wherein thecarrier matrix is human insoluble collagenous bone matrix (hICBM). 41.The bone growth inducing composition as claimed in claim 38, wherein thegelatine is human gelatine.
 42. The bone growth inducing composition asclaimed in claim 38, wherein the carrier matrix is hICBM, the gelatineis human gelatine and the mass ratio between the osteogenic protein, thehICBM and the gelatine is in the range of 0.4-0.6:800-1200:100-1000. 43.The bone growth inducing composition as claimed in claim 42, wherein themass ratio is about 0.5:1000:200.
 44. A device for inducing bone growthin a mammal, comprising a syringe containing a bone growth inducingcomposition as claimed in claim
 38. 45. A method of inducing boneformation in a mammal having a skeletal defect, comprisingreconstituting a bone growth inducing composition as claimed in claim 38and implanting the reconstituted composition into the skeletal defect ofthe mammal.
 46. A method of inducing the growth of ectopic bone in amammal, comprising reconstituting a bone growth inducing composition asclaimed in claim 38 and implanting the reconstituted composition in anon-bony site of the mammal.
 47. A method of accelerating allogeneicbone healing in a mammal, comprising reconstituting a bone growthinducing composition as claimed in claim 38 and implanting allogeneicbone material together with the reconstituted composition into a site inwhich allogeneic bone healing in the mammal is required.
 48. The methodas claimed in claim 47, wherein the allogeneic bone material is selectedfrom the group consisting of human cortical bone chips, cancellous boneblocks, cancellous bone powder, whole bone and demineralized bonematrix.
 49. A method of accelerating autogenous bone graft healing in amammal, comprising reconstituting a bone growth inducing composition asclaimed in claim 38 and implanting autogenous bone material togetherwith the reconstituted composition into a site in which autogenous bonegraft healing in the mammal is required.
 50. The method as claimed inclaim 49, wherein the autogenous bone material is morselized iliac crestautogenous bone.